Reduced FOXO1 expression accelerates skin wound healing and attenuates scarring.

The forkhead box O (FOXO) family has been extensively investigated in aging and metabolism, but its role in tissue-repair processes remains largely unknown. Herein, we clarify the molecular aspect of the FOXO family in skin wound healing. We demonstrated that Foxo1 and Foxo3a were both up-regulated during murine skin wound healing. Partial knockout of Foxo1 in Foxo1(+/-) mice throughout the body led to accelerated skin wound healing with enhanced keratinocyte migration, reduced granulation tissue formation, and decreased collagen density, accompanied by an attenuated inflammatory response, but we observed no wound phenotype in Foxo3a(-/-) mice. Fibroblast growth factor 2, adiponectin, and notch1 genes were significantly increased at wound sites in Foxo1(+/-) mice, along with markedly altered extracellular signal-regulated kinase 1/2 and AKT phosphorylation. Similarly, transient knockdown of Foxo1 at the wound site by local delivery of antisense oligodeoxynucleotides enhanced skin wound healing. The link between FOXO1 and scarring extends to patients, in particular keloid scars, where we see FOXO1 expression markedly increased in fibroblasts and inflammatory cells within the otherwise normal dermis. This occurs in the immediate vicinity of the keloid by comparison to the center of the mature keloid, indicating that FOXO1 is associated with the overgrowth of this fibrotic response into adjacent normal skin. Overall, our data indicate that molecular targeting of FOXO1 may improve the quality of healing and reduce pathological scarring.

NPY antagonism reduces adiposity and attenuates age-related imbalance of adipose tissue metabolism.

An orexigenic hormone, neuropeptide Y (NPY), plays a role not only in the hypothalamic regulation of appetite, but also in the peripheral regulation of lipid metabolism. However, the intracellular mechanisms triggered by NPY to regulate lipid metabolism are poorly understood. Here we report that NPY deficiency reduces white adipose tissue (WAT) mass and ameliorates the age-related imbalance of adipose tissue metabolism in mice. Gene expression involved in adipogenesis/lipogenesis was found to decrease, whereas proteins involved in lipolysis increased in gonadal WAT (gWAT) of NPY-knockout mice. These changes were associated with an activated SIRT1- and PPARγ-mediated pathway. Moreover, the age-related decrease of de novo lipogenesis in gWAT and thermogenesis in inguinal WAT was inhibited by NPY deficiency. Further analysis using 3T3-L1 cells showed that NPY inhibited lipolysis through the Y1 receptor and enhanced lipogenesis following a reduction in cAMP response element-binding protein (CREB) and SIRT1 protein expression. Therefore, NPY appears to act as a key regulator of adipose tissue metabolism via the CREB-SIRT1 signaling pathway. Taken together, NPY deficiency reduces adiposity and ameliorates the age-related imbalance of adipose tissue metabolism, suggesting that antagonism of NPY may be a promising target for drug development to prevent age-related metabolic diseases.

The Role of Neuropeptide Y in Adipocyte-Macrophage Crosstalk during High Fat Diet-Induced Adipose Inflammation and Liver Steatosis.

Obesity is associated with an increased risk of non-alcoholic fatty liver disease (NAFLD), which is initiated by adipocyte-macrophage crosstalk. Among the possible molecules regulating this crosstalk, we focused on neuropeptide Y (NPY), which is known to be involved in hypothalamic appetite and adipose tissue inflammation and metabolism. In this study, the NPY-/- mice showed a marked decrease in body weight and adiposity, and lower free fatty acid and adipose inflammation without food intake alteration during a high fat diet (HFD). Moreover, NPY deficiency increased the thermogenic genes expression in brown adipose tissue. Notably, NPY-mRNA expression was upregulated in macrophages from the HFD mice compared to that from the mice on a standard diet. The NPY-mRNA expression also positively correlated with the liver mass/body weight ratio. NPY deletion alleviated HFD-induced adipose inflammation and liver steatosis. Hence, our findings point toward a novel intracellular mechanism of NPY in the regulation of adipocyte-macrophage crosstalk and highlight NPY antagonism as a promising target for therapeutic approaches against obesity and NAFLD.

Development of a bioassay to screen for chemicals mimicking the anti-aging effects of calorie restriction.

Suppression of the growth hormone/insulin-like growth factor-I pathway in Ames dwarf (DF) mice, and caloric restriction (CR) in normal mice extends lifespan and delays the onset of age-related disorders. In combination, these interventions have an additive effect on lifespan in Ames DF mice. Therefore, common signaling pathways regulated by DF and CR could have additive effects on longevity. In this study, we tried to identity the signaling mechanism and develop a system to assess pro-longevity status in cells and mice. We previously identified genes up-regulated in the liver of DF and CR mice by DNA microarray analysis. Motif analysis of the upstream sequences of those genes revealed four major consensus sequence motifs, which have been named dwarfism and calorie restriction-responsive elements (DFCR-REs). One of the synthesized sequences bound to hepatocyte nuclear factor-4α (HNF-4α), an important transcription factor involved in liver metabolism. Furthermore, using this sequence information, we developed a highly sensitive bioassay to identify chemicals mimicking the anti-aging effects of CR. When the reporter construct, containing an element upstream of a secreted alkaline phosphatase (SEAP) gene, was co-transfected with HNF-4α and its regulator peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α), SEAP activity was increased compared with untransfected controls. Moreover, transient transgenic mice established using this construct showed increased SEAP activity in CR mice compared with ad libitum-fed mice. These data suggest that because of its rapidity, ease of use, and specificity, our bioassay will be more useful than the systems currently employed to screen for CR mimetics, which mimic the beneficial effects of CR. Our system will be particularly useful for high-throughput screening of natural and synthetic candidate molecules.

Modulation of oxidative phosphorylation machinery signifies a prime mode of anti-ageing mechanism of calorie restriction in male rat liver mitochondria.

Mitochondria being the major source and target of reactive oxygen species (ROS) play a crucial role during ageing. We analyzed ageing and calorie restriction (CR)-induced changes in abundance of rat liver mitochondrial proteins to understand key aspects behind the age-retarding mechanism of CR. The combination of blue-native (BN) gel system with fluorescence Difference Gel Electrophoresis (DIGE) facilitated an efficient analysis of soluble and membrane proteins, existing as monomers or multi-protein assemblies. Changes in abundance of specific key subunits of respiratory chain complexes I, IV and V, critical for activity and/or assembly of the complexes were identified. CR lowered complex I assembly and complex IV activity, which is discussed as a molecular mechanism to minimize ROS production at mitochondria. Notably, the antioxidant system was found to be least affected. The GSH:GSSG couple could be depicted as a rapid mean to handle the fluctuations in ROS levels led by reversible metabolic shifts. We evaluated the relative significance of ROS generation against quenching. We also observed parallel and unidirectional changes as effect of ageing and CR, in subunits of ATP synthase, cytochrome P450 and glutathione S-transferase. This is the first report on such 'putatively hormetic' ageing-analogous effects of CR, besides the age-retarding ones.

NPY Deficiency Prevents Postmenopausal Adiposity by Augmenting Estradiol-Mediated Browning.

The orexigenic hormone neuropeptide Y (NPY) plays a pivotal role in the peripheral regulation of fat metabolism. However, the mechanisms underlying the effects of sex on NPY function have not been extensively analyzed. In this study, we examined the effects of NPY deficiency on fat metabolism in male and female mice. Body weight was slightly decreased, whereas white adipose tissue (WAT) mass was significantly decreased as the thermogenic program was upregulated in NPY-/- female mice compared with that in wild-type mice; these factors were not altered in response to NPY deficiency in male mice. Moreover, lack of NPY resulted in an increase in luteinizing hormone (LH) expression in the pituitary gland, with concomitant activation of the estradiol-mediated thermogenic program in inguinal WAT, and alleviated age-related modification of adiposity in female mice. Taken together, these data revealed a novel intracellular mechanism of NPY in the regulation of fat metabolism and highlighted the sexual dimorphism of NPY as a promising target for drug development to reduce postmenopausal adiposity.

Effects of rikkunshito supplementation on resistance to oxidative stress and lifespan in mice.

AIM: Caloric restriction (CR), which limits the caloric intake to 60-70% of ad libitum (AL) amounts in various experimental animals, delays aging and extends the lifespan. We previously showed that neuropeptide Y (NPY), an appetite-stimulating peptide, is essential for the anti-oxidative and life-extending effects of CR. Here, we investigated whether a Japanese traditional herbal medicine, rikkunshito (RKT), which induces NPY activation, has CR-like life-extending effects. METHODS: First, we evaluated the life-extending activity of RKT by examining the effect of long-term RKT administration on wild-type and NPY knockout mice. Furthermore, we tested whether RKT enhances CR-mediated beneficial effects under AL conditions with a normal diet and under mild CR conditions with a high-fat diet. We then used 3-nitropropionic acid or doxorubicin to induce oxidative stress, and analyzed the differences in survival rate, weight loss, gene expression and cellular oxidative damage among groups. RESULTS: RKT administration did not extend the lifespan of wild-type or NPY knockout mice. In the oxidative stress models, RKT treatment upregulated anti-oxidative gene expression in the liver. Furthermore, RKT administration reduced the oxidative damage in the liver compared to the CR conditions alone. However, on induction of oxidative stress by 3-nitropropionic acid or doxorubicin, RKT administration did not affect the survival rate. CONCLUSIONS: These results show that RKT administration only partially mimics the effects of CR at the cellular level, but not at the organismal level to increase the lifespan of mice. Geriatr Gerontol Int 2019; ••: ••-••.

Mechanisms of Calorie Restriction: A Review of Genes Required for the Life-Extending and Tumor-Inhibiting Effects of Calorie Restriction.

This review focuses on mechanisms of calorie restriction (CR), particularly the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis as an evolutionary conserved signal that regulates aging and lifespan, underlying the effects of CR in mammals. Topics include (1) the relation of the GH-IGF-1 signal with chronic low-level inflammation as one of the possible causative factors of aging, that is, inflammaging, (2) the isoform specificity of the forkhead box protein O (FoxO) transcription factors in CR-mediated regulation of cancer and lifespan, (3) the role for FoxO1 in the tumor-inhibiting effect of CR, (4) pleiotropic roles for FoxO1 in the regulation of disorders, and (5) sirtuin (Sirt) as a molecule upstream of FoxO. From the evolutionary view, the necessity of neuropeptide Y (Npy) for the effects of CR and the pleiotropic roles for Npy in life stages are also emphasized. Genes for mediating the effects of CR and regulating aging are context-dependent, particularly depending on nutritional states.

Pituitary growth hormone suppression reduces resistin expression and enhances insulin effectiveness: relationship with caloric restriction.

Caloric restriction (CR) retards various age-dependent disorders, increases lifespan, and improves insulin activity in laboratory animals. Recently, adipocytes were found to act together as an active endocrine organ that produces various hormones called adipocytokines. The peripheral and central activities of these adipocytokines have been suggested to mediate the anti-aging effects of CR. Here, we tested this notion by analyzing the effect of CR and suppression of growth hormone/insulin-like growth factor-I (GH/IGF-I) axis on the expression of resistin, adiponectin, and adipsin genes by rat white adipose tissue (WAT). We found that CR and GH/IGF-I suppression markedly downregulated resistin gene expression. We also found plasma resistin levels correlated positively with pituitary GH mRNA expression levels. Our observations suggest that CR reduces resistin expression and increases insulin effectiveness in a GH/IGF-I-dependent manner. The beneficial effects of CR and GH/IGF-I suppression appear to be mediated, at least in part, by changes in glucose metabolism that result from reductions in plasma resistin levels.

Manipulation of caloric content but not diet composition, attenuates the deficit in learning and memory of senescence-accelerated mouse strain P8.

Calorie restriction (CR) is an experimental intervention in laboratory animals that attenuates age-associated increases in morbidity, mortality, and functional impairment. It is characterized by mild ketosis, hypoinsulinemia and hypoglycemia. In this study, we examined whether metabolic simulation of CR by a diet of isocaloric ketogenic or hypoinsulinemic diets ameliorated the learning and memory deficit in a strain of senescence-accelerated prone mice (SAMP8), a mouse model of age-dependent impairments in learning and memory. Male SAMP8 mice were fed high carbohydrate (CHO), high fat (FAT), or high protein (PRO) diets after weaning, and calorie intake was adjusted to 95% (sub ad libitum, sAL) or 70% (CR) of the mean calorie intake of control mice. At 28 weeks of age, we found CR ameliorated the performance defects of SAMP8 mice in a passive avoidance task. Neither FAT nor PRO diets affected performance of the task when fed sAL level, although a diet of these compositions partially mimicked the serum parameters of CR mice. These results suggest restriction of calorie intake is important for the prevention of learning and memory deficits, and that the simulation of serum changes induced by CR is not sufficient to prevent the cognitive defects of SAMP8 mice.

Longevity genes: insights from calorie restriction and genetic longevity models.

In this review, we discuss the genes and the related signal pathways that regulate aging and longevity by reviewing recent findings of genetic longevity models in rodents in reference to findings with lower organisms. We also paid special attention to the genes and signals mediating the effects of calorie restriction (CR), a powerful intervention that slows the aging process and extends the lifespan in a range of organisms. An evolutionary view emphasizes the roles of nutrient-sensing and neuroendocrine adaptation to food shortage as the mechanisms underlying the effects of CR. Genetic and non-genetic interventions without CR suggest a role for single or combined hormonal signals that partly mediate the effect of CR. Longevity genes fall into two categories, genes relevant to nutrient-sensing systems and those associated with mitochondrial function or redox regulation. In mammals, disrupted or reduced growth hormone (GH)-insulin-like growth factor (IGF)-1 signaling robustly favors longevity. CR also suppresses the GH-IGF-1 axis, indicating the importance of this signal pathway. Surprisingly, there are very few longevity models to evaluate the enhanced anti-oxidative mechanism, while there is substantial evidence supporting the oxidative stress and damage theory of aging. Either increased or reduced mitochondrial function may extend the lifespan. The role of redox regulation and mitochondrial function in CR remains to be elucidated.

Targeting miR-223 in neutrophils enhances the clearance of Staphylococcus aureus in infected wounds.

Argonaute 2 bound mature microRNA (Ago2-miRNA) complexes are key regulators of the wound inflammatory response and function in the translational processing of target mRNAs. In this study, we identified four wound inflammation-related Ago2-miRNAs (miR-139-5p, miR-142-3p, miR-142-5p, and miR-223) and show that miR-223 is critical for infection control. miR-223Y/- mice exhibited delayed sterile healing with prolonged neutrophil activation and interleukin-6 expression, and markedly improved repair of Staphylococcus aureus-infected wounds. We also showed that the expression of miR-223 was regulated by CCAAT/enhancer binding protein alpha in human neutrophils after exposure to S. aureus peptides. Treatment with miR-223Y/--derived neutrophils, or miR-223 antisense oligodeoxynucleotides in S. aureus-infected wild-type wounds markedly improved the healing of these otherwise chronic, slow healing wounds. This study reveals how miR-223 regulates the bactericidal capacity of neutrophils at wound sites and indicates that targeting miR-223 might be of therapeutic benefit for infected wounds in the clinic.

Down-regulation of AMP-activated protein kinase by calorie restriction in rat liver.

AMP-activated protein kinase (AMPK) may act as a key enzyme for metabolic adaptation to calorie restriction (CR) or reduced growth hormone (GH)-insulin-like growth factor (IGF)-1 signaling, an experimental intervention for lifespan extension in animals. We investigated the protein levels of AMPKalpha and a downstream enzyme, acetyl-CoA carboxylase (ACC), by immunoblotting of liver and quadriceps femoris muscle (QFM) extracts from 6-month-old wild-type (W) and GH-suppressed transgenic (Tg) Wistar rats fed ad libitum (AL) or 30% CR diets from 6weeks of age. A modified alternate-day feeding regimen for CR yielded a fed-fasted cycle in CR rats, and therefore the effects of overnight fasting in W-AL rats were also evaluated. CR decreased threonine-172-phosphorylated AMPKalpha (p-AMPKalpha; an activated form) levels in the liver, whereas the CR-fed-fasted cycle or overnight fasting did not significantly affect the p-AMPKalpha level. In the QFM, the p-AMPKalpha level was slightly elevated in the CR-fasted phase, but greatly increased in the AL-fasted phase. Suppression of GH did not affect the p-AMPKalpha level. The phosphorylated-ACC levels did not alter in parallel with the p-AMPKalpha level, particularly in the liver. The present results suggest that CR down-regulates the AMPK activity in the liver on a long-term basis.

Identification of fasting-induced genes in the rat hypothalamus: relationship with neuroprotection.

During food shortage, organisms activate defense mechanisms to maximize their chance of survival. At least in part, these responses are triggered by changes in hormonal status and neural status during starvation. The hypothalamus is organized as a collection of distinct autonomously active nuclei and is considered to play crucial roles in these survival responses. To isolate factors involved in these pathways, we carried out suppression subtractive hybridization analyses using complementary DNAs (cDNA) from the hypothalami of fasted and fed rats. We identified four genes, namely ubiquitin-conjugating enzyme E2D 3 (UBE2D3), cAMP-dependent protein kinase C beta subunit (PKCbeta), excitatory amino acid carrier 1 (EAAC1), and ferritin heavy polypeptide 1 (Fth1), that were upregulated after a 48-h fast compared to the fed status. According to previous reports, these genes have been implicated in protection against neuronal cell death under various neurodegenerative stresses, such as hypoxia-ischemia and oxidative stress. Thus, the increased expressions of the genes identified in the present study may have protective effects against neural damage that could otherwise result in cell death.

Involvement of insulin-like growth factor-1 in the effect of caloric restriction: regulation of plasma adiponectin and leptin.

Insulin-like growth factor (IGF)-1 signaling might partly mediate effects of caloric restriction (CR), an experimental intervention for increasing longevity in mammals. The present study evaluated effects of recombinant human (rh)IGF-1 infusion on adipokine levels in CR and transgenic (Tg) dwarf rats with the reduced growth hormone-IGF-1 axis, which shared similar body weight and food intake. At 9 months of age, each rat received a continuous infusion of rhIGF-1 for 14 days, and rats received an injection of glucose after overnight fasting. Infusion of rhIGF-1 had metabolic effects in all rat groups although it did not affect insulin sensitivity in any of the groups. In addition, plasma adiponectin was decreased to the control group levels and plasma leptin was further reduced in CR and Tg rats. The similarity of phenotypes and adipokine responses to rhIGF-1 between CR and Tg rats supports a role for reduced IGF-1 signaling in the CR effect.

Neuropeptide Y resists excess loss of fat by lipolysis in calorie-restricted mice: a trait potential for the life-extending effect of calorie restriction.

Neuropeptide Y (NPY) is an orexigenic peptide that plays an essential role in caloric restriction (CR)-mediated lifespan extension. However, the mechanisms underlying the NPY-mediated effects in CR are poorly defined. Here, we report that NPY deficiency in male mice during CR increases mortality in association with lipodystrophy. NPY-/- mice displayed a rapid decrease in body weight and fat mass, as well as increased lipolysis during CR. These alterations in fat regulation were inhibited by the lipolysis inhibitor, acipimox, a treatment associated with reduced mortality. The lipolytic/thermogenic signaling, ß3-adrenergic receptor/hormone sensitive lipase, was markedly activated in white adipose tissue of NPY-/- mice compared with that of NPY+/+ mice, and thermogenesis was controlled by NPY under negative energy balance. These results demonstrate the critical role of NPY in the regulation of lipid metabolic homeostasis and survival via control of lipolysis and thermogenesis in a state of negative energy balance.

MiR-142 Is Required for Staphylococcus aureus Clearance at Skin Wound Sites via Small GTPase-Mediated Regulation of the Neutrophil Actin Cytoskeleton.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate protein translation by binding to complementary target mRNAs. We previously identified two mature members of the miR-142 family, miR-142-5p and miR-142-3p, as inflammation-related miRNAs with potential roles in wound healing. Here, we demonstrated that these two miRNAs are prominently expressed in wound-infiltrated neutrophils and macrophages and play central roles in wound healing. We generated miR-142-/- mice using the exchangeable gene-trap method and showed that healing of Staphylococcus aureus-infected skin wounds was significantly delayed in miR-142-/- mice compared with that in wild-type mice. MiR-142-/- mice exhibited abnormal abscess formation at S. aureus-infected skin wound sites and were also more susceptible to horizontal transmission of wound infections. MiR-142-/- neutrophils showed altered phagocytosis as a consequence of chemotactic behavior, including enhanced F-actin assembly, disturbed cell polarity, and increased cell motility. We showed that these changes were linked to cytoskeletal regulation, and that expression of the small GTPases was markedly increased in miR-142-/- neutrophils. Collectively, our data demonstrate that the miR-142 family is indispensable for protection against S. aureus infection and its clearance at wound sites. MiR-142-3p and miR-142-5p play nonredundant roles in actin cytoskeleton regulation by controlling small GTPase translation in neutrophils at wound sites.

Calorie restriction initiated at middle age improved glucose tolerance without affecting age-related impairments of insulin signaling in rat skeletal muscle.

Calorie restriction (CR) may affect glucose tolerance via modulation of the insulin action in skeletal muscle. The present study investigated the effect of CR initiated at middle age in rats bearing glucose intolerance, in comparison with CR at a younger age. Male F344 rats at 2.5 and 18months (mo) of age were fed ad libitum (AL) or 30% CR diets for 4-4.5mo, subjected to glucose tolerance testing, and then sacrificed 15min after intraperitoneal glucose or saline injection to evaluate glucose-stimulated insulin response and subsequent activation of insulin signaling molecules. The protein abundance of phosphorylated (p) insulin receptors, p-Akt, and p-atypical PKC and the membrane fraction of glucose transporter 4 in quadriceps femoris muscle (QFM) were analyzed by EIA or immunoblotting. CR initiated either at young or middle age improved glucose tolerance with a lower serum insulin response to glucose. However, middle-aged CR did not improve aging-related impairments in insulin signaling in QFM. The present results emphasized the possibilities of CR activation of an insulin-independent mechanism in skeletal muscle and also of the involvement of non-muscle tissues in glucose uptake.

Hepatic gene expression profile of lipid metabolism in rats: Impact of caloric restriction and growth hormone/insulin-like growth factor-1 suppression.

We investigated the role of the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis on caloric restriction (CR) using male wild-type and transgenic homozygous dwarf rats bearing an antisense GH transgene and their F1 heterozygous progeny fed either ad libitum or subjected to 30% CR. CR predominantly altered expression of hepatic genes involved in the stress response, xenobiotic metabolism, and lipid metabolism. Most gene expressions involved in stress response and xenobiotic metabolism were regulated in a GH/IGF-1-dependent manner, and those involved in lipid metabolism were regulated in a GH/IGF-1-independent manner. Moreover, CR enhanced the gene expression involved in fatty acid synthesis after feeding and those encoding mitochondrial beta-oxidation enzymes during food shortage, probably via transcriptional regulation by peroxisome proliferator-activated receptor alpha. These results, taken together with serum biochemical measures and hepatic triglyceride content, suggest that CR promotes lipid utilization through hepatic transcriptional alteration and prevents hepatic steatosis in a GH/IGF-1-independent manner.

Effect of leptin on hypothalamic gene expression in calorie-restricted rats.

Diminished leptin signaling to the arcuate nucleus of hypothalamus (ARH) may induce calorie restriction (CR)-specific neuroendocrine and metabolic adaptation, which is potentially relevant to the effect of CR. The present study investigated whether restoration of leptin signaling to the ARH could reverse CR-induced alterations in neuropeptide gene expression in rats. Male F344 rats, fed ad libitum or a 30% CR diet from 6 weeks of age, received leptin or vehicle intracerebroventricularly for 14 days via osmotic mini-pumps implanted in the subcutis at 34 weeks of age. The messenger RNA levels were quantified by real-time reverse transcription-polymerase chain reaction using total RNA extracted from microdissected tissues containing the ARH. The results indicated that leptin administration reversed the upregulated expression of neuropeptide Y and agouti-related protein genes in CR rats, suggesting the possibility of a role for the leptin-ARH pathway in the effect of CR.